DISH PROTOCOLS
1) WHEN IN DOUBT, THROW IT OUT! If you don't know for sure something is sterile, it isn't sterile.
2) A flask or DT on the shelf is sterile. DO NOT EVER put anything on the flask/DT shelves unless
they have just been taken out of the autoclave!
3) NEVER put a flask or DT back on the sterile shelf. Once you take it, it's yours.
4) Nothing goes in the acid-washed cabinet unless it has gone through the full acid-wash cycle. Nothing
goes back in the acid-washed cabinet after it has been taken out.
5) 28 flasks will fit in one Lenski flask tray! Fill them up to save space.
6) ALL CONTAMINATED DISHES go in the space to the right of the microwave oven/fume hood.
NO DISHES to the left of the flow cytometer are clean!
FLASK PROTOCOL
1) To grow cultures in Lenski flasks, take the required number of flasks from the LEFT SIDE of the
flask shelf. Take only as many as you need and do not put any back.
2) REMOVE AUTOCLAVE TAPE from Lenski flask trays when you remove them from the flask
shelf!!!
3) Check your flask for excessive residue or cracks before adding media.
4) When you are done with a culture in a Lenski flask:
 Remove any labels using 100% ethanol
 Add water to ~20 mL (to prevent media from drying in the autoclave and leaving residue)
 Place flask in a stainless steel Lenski flask tray with a fresh piece of autoclave tape on the
corner to indicate that the flasks in the tray are dirty.
5) If you need to keep cultures after removing them from the autoclave, put them in a tray in the fridge
or in the cold room. Do not keep them on your bench! There's no point; after sitting overnight at
room temperature you can't use them anymore.
6) Every morning, dirty flasks should be autoclaved in their Lenski flask trays. Use liquid cycle, 30
minutes. Autoclave dirty flasks/DTs separately from fresh media.
7) After coming out of the autoclave, pour contents down drain and soak flasks and beakers in Micro-90
detergent for ~ 1 hour. Check for any remaining residue and if necessary clean with a wire brush.
8) If flasks can't be washed immediately after autoclaving, Lenski flask trays with used autoclave tape
still on should be stacked to the left of the dish sink. NO CONTAMINATED DISHES TO THE
RIGHT OF THE FLOW CYTOMETER!
9) Rinse flasks with deionized water 3x. Assemble with washed beakers in Lenski flask trays. No need
to let them dry.
10) Place autoclave tape on the corner of each tray of clean flasks. Autoclave on the gravity cycle, 20
minutes.
11) Slide old trays of clean flasks to the left side of the flask shelf. Add newly autoclaved trays to the
RIGHT SIDE of the flask shelf.
DT PROTOCOL
1) To make sterile saline for Dilution Tubes, dissolve 8.5 g of NaCl in 1 L of milli-Q water and
autoclave on liquid cycle for 30 minutes.
2) Sterilize DT tubes (16x150 mm glass) on the gravity cycle for 20 minutes. Put autoclave tape on the
front of the tube rack, not on the tubes themselves.
3) DT's each contain 9.9 mL of saline. Working in the hood, use either a 10 mL sterile pipet or else a
bottle-top pump to dispense from autoclaved saline bottle into autoclaved tubes.
4) Move old DT's to the left side of the DT shelf and add newly made ones to the RIGHT side of the
shelf.
5) To use DT's, take just what you need from the LEFT side of the DT shelf. Never put DT's back on
the DT shelf!
6) If you take the last DT from a tube rack on the DT shelf, TAKE THE AUTOCLAVE TAPE OFF
THE RACK!
7) When done with DT's, remove labels with 100% ethanol and put in a tube rack with a fresh piece of
autoclave tape on the front.
8) Every morning, dirty DT's should be autoclaved. Use liquid cycle, 30 minutes. Autoclave dirty
flasks/DTs separately from fresh media.
9) After coming out of the autoclave, pour contents down drain and soak tubes and caps in Micro-90
detergent for ~ 1 hour. Check for any remaining residue and if necessary clean with a wire brush.
10) If tubes can't be washed immediately after autoclaving, racks with used autoclave tape still on should
be stacked to the left of the dish sink. NO CONTAMINATED DISHES TO THE RIGHT OF THE
FLOW CYTOMETER!
11) Rinse tubes with deionized water 3x. Assemble with washed caps and proceed with step 2.
MISCELLANEOUS GLASSWARE PROTOCOL
1) Miscellaneous glassware (flasks, bottles, graduated cylinders, etc) is washed by soaking for at least 1
hour in Micro-90 detergent followed by 3 rinses with deionized water.
2) Most glassware should be dried by either hanging on the racks above the sinks or else in the plastic
dish-drying rack.
3) Used glassware that has NOT been contaminated with microbes should NOT be placed to the left of
the flow cytometer for autoclaving. These dishes should be emptied and placed in the sink instead.
4) Do not pour chemicals down the drain without checking to make sure it's okay!
5) Make sure all labels are removed from dishes before they go in the sink.
6) To label glassware, use LABEL TAPE -- it comes off much easier than "normal" tape.
STIR BAR PROTOCOL: Wash stir bars like other dishes, but make sure not to dump them down the
drains when rinsing! Clean stir bars should be placed in a beaker near the media prep area.